Marina Marchetti

Venous thromboembolism in the hematologic malignancies

Patients with hematologic malignancies are at high risk of thrombotic or hemorrhagic complications. The incidence of these events is greatly variable and is influenced by many factors, including the type of disease, the type of chemotherapy, and the use of a central venous device. As in solid tumors, a number of clinical risk factors have been identified and contribute to the increasing thrombotic rate in hematologic malignancies. Biologic properties of the tumor cells can influence the hypercoagulable state of patients with these malignancies by several mechanisms. Of interest, oncogenes responsible for neoplastic transformation in leukemia also may be involved in clotting activation. Epidemiologic data allow an estimate of the incidence of venous thromboembolism (VTE) in acute leukemia, lymphomas, and multiple myeloma (MM). In this review, we focus on the epidemiology, pathogenesis, and VTE management in these three hematologic malignancies. No recommendation for routine thromboprophylaxis in these conditions, with the exception of MM, is available. Large, prospective, randomized clinical trials are needed to establish the best practice for thromboprophylaxis and treatment of VTE in these types of cancers.

Coagulation and cancer: biological and clinical aspects.

Summary.  Malignancy affects the hemostatic system and the hemostatic system affects malignancy. In cancer patients there are a number of coagulation abnormalities which provide the background for an increased tendency of these patients to both thrombosis and hemorrhage. The causes of this coagulation impairment rely on general risk factors which are common to other categories of patients, and other factors which are specific to cancer, such as tumor type and disease stage. In addition, data from basic research indicate that the hemostatic components and the cancer biology are interconnected in multiple ways. Notably, while cancer cells are able to activate the coagulation system, the hemostatic factors play a role in tumor progression. This opens the way to the development of bifunctional therapeutic approaches that are both capable of attacking the malignant process and resolving the coagulation impairment. On the other hand, the management of thrombosis and hemorrhages in cancer patients can be different. To approach these problems, some guidelines have been released by prominent international scientific societies. Also actively investigated is the issue of identifying new biomarkers to classify the subjects at a higher risk, thus improving the prevention of thrombohemorrhagic events in these patients. Finally, novel prophylactic and therapeutic approaches are currently under development. This review provides an overview of the hemostatic complications in cancer, together with new insights into the interaction between hemostasis and cancer biology. We also review the assessment of the risk of thrombohemorrhagic events in cancer patients, and the prophylaxis and treatment of such manifestations.

Thrombin generation and activated protein C resistance in patients with essential thrombocythemia and polycythemia vera

We used the thrombin generation assay to evaluate the hypercoagulable state according to JAK2(V617F) mutational status in essential thrombocythemia (ET) and polycythemia vera (PV) patients. Thrombin generation was determined in the presence and absence of activated protein C (APC), and APC resistance was expressed as normalized APC sensitivity ratio (nAPCsr). Tissue factor pathway inhibitor (TFPI), total and free protein S (PS), prothrombin (FII), factor V (FV), and neutrophil elastase were measured in plasma; CD11b was measured on neutrophils. Compared with normal controls, patients had a lower endogenous thrombin potential in the absence of APC but had a higher endogenous thrombin potential in the presence of APC, showing the occurrence of APC resistance. The nAPCsr increased in JAK2(V617F) carriers compared with noncarriers and was highest in JAK2(V617F) homozygous patients. FII, FV, free PS, and TFPI levels were reduced in patients, mainly in JAK2(V617F) carriers. Multiple regression analysis indicated the low free PS level as major determinant of the increased nAPCsr. Elastase was increased in patients and inversely correlated with free PS. In conclusion, these data indicate the occurrence of acquired APC resistance in ET and PV patients, probably because of a reduction in free PS levels. The APC-resistant phenotype is influenced by the JAK2(V617F) mutational load.

Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia

Essential thrombocythemia is a myeloproliferative neoplasm characterized by an increased risk of both arterial and venous thrombosis. The findings of this study show that patients with this disorder have elevated levels of platelet-and endothelium-derived microparticles, which may support thrombin generation and play a role in the pathophysiology of thromboembolic complications. Background Most cell types, including blood - and vascular cells, produce microparticles upon activation. Since cellular microparticles are known to be elevated in thromboembolic diseases, we hypothesized a role for microparticles in the pathogenesis of thrombosis in essential thrombocythemia. Design and Methods In plasma samples from 21 patients with essential thrombocythemia and ten healthy subjects, the levels and the cellular origin of microparticles were determined by flowcytometric analysis, while the microparticle-associated procoagulant activity was measured using a thrombin generation assay. Results Patients with essential thrombocythemia had significantly higher numbers of circulating annexin V-positive microparticles than controls (median 4500 vs. 2500×106 events/L; p=0.039), including significantly higher numbers of microparticles positive for the platelet marker CD61 (p=0.043), the endothelial markers CD62E (p=0.009) and CD144 (p=0.021), and for tissue factor (p=0.036). CD62E was co-expressed with the platelet marker CD41 on microparticles, suggesting a bilineage origin of such microparticles, which were observed only in patients with risk factors for thrombosis. Patients with essential thrombocythemia had higher plasma levels of mature von Willebrand factor (p=0.045) but similar propeptide levels compared to controls. In thrombin generation analyses, microparticle-rich plasma from patients with essential thrombocythemia had a shorter lag time (p=0.001) and higher peak height (p=0.038) than plasma from controls. Peak height correlated significantly with the total number of microparticles (R=0.634, p<0.001). Conclusions Patients with essential thrombocythemia had higher number of circulating microparticles with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This was confirmed by an increased level of mature von Willebrand factor, an abnormal mature von Willebrand factor/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for microparticles in thrombosis in essential thrombocythemia.

Thrombotic disease in the myeloproliferative neoplasms

Thrombosis is a leading cause of morbidity and mortality in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), particularly polycythemia vera and essential thrombocythemia. Mechanisms involved in the pathogenesis of the acquired thrombophilic state associated with these diseases include abnormalities of MPN clone-derived blood cells, which display prothrombotic features, and abnormalities of normal vascular cells, which become procoagulant in response to inflammatory stimuli. Ultimately, the release into the blood of elevated levels of procoagulant microparticles by platelets and vascular cells and the increase in the global thrombin generation due to an acquired activated protein C resistance result in a highly prothrombotic scenario in patients with polycythemia vera and essential thrombocythemia. The acquired point mutation in the pseudokinase domain of JAK2 (JAK2V617F) in these disorders is variably associated with thrombosis and, more consistently, with elevations in WBC counts and alterations in biomarkers of blood-clotting abnormalities. The predictive value of these biomarkers for thrombosis remains to be established to identify subsets of patients at elevated risk who may benefit from prophylaxis with antithrombotic drugs.

Platelet-induced thrombin generation by the calibrated automated thrombogram assay is increased in patients with essential thrombocythemia and polycythemia vera.

The platelet contribution to the thrombophilic state of patients with myeloproliferative neoplasms (MPNs), i.e., essential thrombocythemia (ET) and polycythemia vera (PV), remains uncertain. In this study we aimed to characterize the thrombin generation (TG) potential expressed by platelets from these subjects, compare it to normal platelets, and identify what factors might be responsible for platelet TG. In a group of 140 MPN patients (80 ET and 60 PV) and 72 healthy subjects we measured the global procoagulant potential of platelet rich plasma (PRP) utilizing the TG assay by the calibrated automated thrombogram (CAT). To characterize the procoagulant contribution of platelets in PRP, the TG of both isolated platelets and platelet poor plasma was measured, and the platelet surface expression of TF was determined. Finally, the activation status of platelets was assessed by the levels of P‐selectin expressed on platelet surface. MPN patients had significantly increased PRP and isolated platelet TG potential compared to controls. This was associated to the occurrence of platelet activation. Patients carriers of the JAK2V617F mutation showed the highest values of TG and platelet surface TF and P‐selectin. Platelet TG potential was significantly lower in hydroxyurea (HU) compared to non‐HU‐treated patients and was lowest in HU‐treated JAK2V617F carriers. In subjects not receiving HU, platelet TG significantly increased by JAK2V617F allele burden increment (P < 0.05). This study demonstrates a platelet‐dependent form of hypercoagulability in MPN patients, particularly in those carriers of the JAK2V617F mutation. The cytoreductive therapy with HU significantly affects this prothrombotic phenotype. Am. J. Hematol. 86:337–342, 2011.

Prospective study of hemostatic alterations in children with acute lymphoblastic leukemia

In a group of newly diagnosed acute lymphocytic leukemia (ALL) children we evaluated a number of hemostatic and inflammatory markers at diagnosis and at different time points during chemotherapy for the remission induction to identify alterations in the plasma levels of prothrombotic markers before and during the course of chemotherapy. The following plasma markers were evaluated: thrombin‐antithrombin complex (TAT), D‐Dimer, plasminogen activator inhibitor 1 (PAI‐1), antithrombin, fibrinogen, von Willebrand factor (VWF) antigen and high molecular weight VWF (HMW‐VWF) multimers, P‐selectin, tumor necrosis factor alpha (TNF‐α), and interleukin 6 (IL‐6). Plasma samples were collected at the following time points: at T0 (baseline) and T1 (+24 days of therapy), T2 (+36 days therapy), and T3 (+64 days therapy). The results show that, at diagnosis, ALL children presented with laboratory signs of increased thrombin generation and fibrin formation (i.e. high TAT and D‐dimer levels), fibrinolysis inhibition (i.e. high PAI‐1 level), endothelial activation (i.e., high HMW‐VWF and soluble P‐selectin levels) and inflammation (i.e. high TNF‐alpha and IL‐6 levels). After starting induction therapy, the thrombin generation markers and inflammatory cytokines significantly decreased. To the opposite, PAI‐1 and P‐selectin significantly increased, suggesting an insult by chemotherapy on the vascular endothelium. These effects were more evident during steroid administration. Symptomatic venous thromboembolism (VTE) episodes developed in two cases during induction therapy, which did not allow the evaluation of the predictive value for VTE of laboratory markers. Am. J. Hematol., 2010.

JAK2V617F mutation and hydroxyurea treatment as determinants of immature platelet parameters in essential thrombocythemia and polycythemia vera patients

Immature platelets (IPFs), which are hemostatically more active than mature platelets, have been found elevated in essential thrombocythemia and polycythemia vera, 2 myeloproliferative neoplasms (MPN) characterized by an increased risk of thrombosis. It is not known whether the IPF levels are influenced by pathogenetic factors, including JAK2V617F mutational status, or by treatment regimen. To address this point, in 46 essential thrombocythemia and 38 polycythemia vera consecutive patients, we measured IPF and correlated the results to JAK2V617F mutation and myelosuppressive treatment with hydroxyurea. This analysis provides 2 new elements regarding IPF and MPN. The first finding is that the JAK2V617F mutation is linked to the quantity of IPF in patients with MPN, which might contribute to the prothrombotic phenotype in these patients. The second finding is that IPF is susceptible to myelosuppressive treatment, which may additionally explain the favorable effect of hydroxyurea therapy on MPN outcome as well as the associated thrombotic risk.

The mechanisms of cancer-associated thrombosis.

Patients with cancer may display many types of hemostatic disorders that significantly contribute to morbidity and mortality in this disease. A complex coagulopathy develops in parallel with malignancy and is characterized by activation of clotting mechanisms to different extent in different patients and in different types of tumor. The pathogenesis of hemostatic alterations in cancer is multifactorial; however, the tumor tissue capacity to interact with and activate the host hemostatic system plays an important role. New molecular pathways of regulation of these properties have been recently demonstrated. Intervention strategies to prevent and treat venous thromboembolism (VTE) in cancer patients have been addressed by large RCTs and guidelines for VTE management have been updated. In this review, we will present an updated overview of the complex coagulopathy associated to malignancy and of recent advances in the thrombotic risk assessment of cancer patients.

Microparticles in tumor progression

Microparticles (MP) are shed from the surface of activated or apoptotic blood cells and their levels in plasma reflect a balance between cell stimulation, proliferation, and death. MP production occurs through vesiculation of cell membranes, and involves cytoskeletal changes and a shift in the normal phospholipid asymmetry. The expression on the majority of MP of the anionic phosphatidylserine (PS) is responsible for the capacity of MP to support blood coagulation activation. In some cases, PS expression is also associated, in the same MP, with the presence of active Tissue Factor, the main activator of blood coagulation. Elevation in plasma levels of MP have been described in numerous clinical conditions, most of which also associated with an increased thrombotic risk. Particularly, MP have been found to be increased in both solid and hematological malignancies, including myeloproliferative neoplasms. A role of MP in tumor progression has been suggested by both in vitro and in vivo studies. Evidence exists that MP of platelet origin are the main players in this process, being rich in pro-angiogenic factors. The utility of measuring MP as a diagnostic and prognostic marker is currently a subject of intense investigation. The possibility to inhibit MP production by pharmacological interventions represents a future challenge.